Incorporation of nucleoside triphosphates (dNTPs) labeled with fluorescent dyes is important for many in vitro DNA synthesis applications. For example, dye-terminator DNA sequencing reactions require the incorporation of fluorescent dideoxynucleotide analogues for termination and labeling. In addition, in vitro synthesis of labeled products may involve incorporation of fluorescent nucleotides or nucleotide analogues. For example, fluorescently labeled DNA has been used in hybridization assays using microarrays of immobilized probes (Cronin et al., 1996, Human Mutation 7:244).
To assure fidelity of DNA replication, DNA polymerases have a very strong bias for incorporation of their normal substrates, referred to herein as conventional deoxynucleoside triphosphates (dNTPs), and against incorporation of unconventional dNTPs including dNTPs and dNTP analogues labeled with fluorescent dyes. In the cell, this property attenuates the incorporation of abnormal bases such as dUTP in a growing DNA strand. In vitro, this characteristic is particularly evident where both conventional and unconventional fluorescently-labeled nucleoside triphosphates are present, such as in DNA sequencing reactions using a version of the dideoxy chain termination method that utilizes dye-terminators (Lee et al., 1992, Nuc. Acids. Res. 20:2471 which is incorporated herein by reference).
Commercially available DNA cycle sequencing kits for dye-terminator methods use chain terminator ddNTPs labeled with fluorescent dyes of the rhodamine family.
However, rhodamine dyes are zwitterionic in charge and nucleoside triphosphates labeled with these dyes migrate anomalously in the electrophoretic gels used to separate the sequencing products for detection. This property of rhodamine family dyes necessitates making modifications in the standard sequencing protocol which include the use of dITP and an additional processing step before electrophoresis.
In contrast, negatively charged fluorescent dyes such as fluorescein family dyes allow 1) better separation between the labeled nucleoside triphosphates and labeled primer extension products, and 2) better electrophoretic migration of the labeled sequencing products than neutral or positively charged fluorescent dyes. Thus, the use of fluorescein family dyes avoids the need for additional processing steps required with the use of rhodamine family dyes. However, available dyes of the fluorescein family are not ideal for use in current commercially available DNA cycle sequencing formats because ddNTPs labeled with these dyes are not efficiently incorporated into sequencing products using these formats. Consequently, there is a need for commercially available thermostable DNA polymerases that can efficiently incorporate both conventional and fluorescein-labeled nucleotides. The present invention serves to meet that need. Further, an unexpected property of the mutant enzymes of this invention is the increased rate of primer extension relative to the corresponding wild-type enzyme. Another unexpected property is the increased uniformity of incorporation of the various terminator nucleotides in automated DNA sequence analysis.